Experimental principle of human 12-hydroxyeicosapentaenoic acid (12-HETE) ELISA

Human 12-hydroxyeicosapentaenoic acid (12-HETE) ELISA test principle This reagent is for research use only: This kit is used to determine 12-hydroxyeicosapentaenoic acid (12 in human serum, plasma and related liquid samples -HETE) content. Experimental principle: This kit uses the double antibody sandwich method to determine the level of 12-HETE in the specimen. Microporous plates were coated with purified human 12-hydroxyeicosapentaenoic acid (12-HETE) antibody to prepare solid-phase antibodies, and 12-hydroxyeicosapentaenoic acid (12- HETE), and then combined with HRP-labeled 12-hydroxyeicosapentaenoic acid (12-HETE) antibody to form an antibody-antigen-enzyme-labeled antibody complex, after thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with 12-hydroxyeicosapentaenoic acid (12-HETE) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human 12-hydroxyeicosapentaenoic acid (12-HETE) in the sample was calculated by a standard curve. Kit composition: kit composition 48 well configuration 96 well configuration storage instructions 1 copy 1 copy sealing film 2 pieces (48) 2 pieces (96) 1 sealed bag 1 enzyme coated plate 1 × 48 1 × 96 2 Store standard at -8 ℃: 135ng / ml 0.5ml × 1 bottle 0.5ml × 1 bottle at 2-8 ℃, store standard dilution 1.5ml × 1 bottle 1.5ml × 1 bottle at 2-8 ℃, store enzyme label reagent 3 ml × 1 bottle of 6 ml × 1 bottle of sample diluent at 2-8 ° C 3 ml × 1 bottle of 6 ml × 1 bottle of 2-8 ° C Developer A 3 ml × 1 bottle of 6 ml × 1 bottle of 2-8 ° C Developer B solution 3 ml × 1 bottle 6 ml × 1 bottle at 2-8 ° C storage stop solution 3ml × 1 bottle 6ml × 1 bottle at 2-8 ° C Concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle at 2-8 ° C to store the sample. Processing and requirements: 1. Serum: room temperature blood is naturally coagulated for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage. 2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation. 4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use. 6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided. 7. Samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP). Operation steps: standard dilution and sample addition: set up 10 standard wells on the enzyme-coated plate, add 100 μl of the standard in the first and second wells, and then add the standard in the first and second wells 50μl of the product dilution, mix well; then take 100μl from the first well and the second well respectively to the third and fourth wells, and then add 50μl of the standard dilution solution to the third and fourth wells respectively, mix well ; Then in the third and fourth wells, first take 50μl to discard, and then take 50μl respectively to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix well ; After mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. Take 50μl from the eighth well and add it to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells respectively. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl, and the concentrations are 90 ng / ml, 60 ng / ml, 30 ng / ml, 15 ng / ml, 7.5 ng / ml). Sample addition: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix. Incubation: Seal the plate with a sealing film and incubate at 37 ° C for 30 minutes. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing solution with distilled water 30 times (20 times of 48T) and then reserve. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let it stand for 30 seconds, then discard, repeat 5 times and pat dry. Enzyme addition: Add 50μl of enzyme label reagent to each well, except for blank wells. Incubation: The operation is the same as 3. Washing: The operation is the same as 5. Color development: Add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes. Termination: Add 50μl of stopper solution to each well to stop the reaction (blue at this time) Li to yellow). Measurement: The absorbance (OD value) of each well was measured in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution. Note: The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before use. If the enzyme label coated plate is unopened after opening, the strip should be stored in a sealed bag. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× n × 5). The sealing film is limited to one-time use to avoid cross-contamination. Please keep the substrate away from light. Carry out the operation in strict accordance with the instructions. The test result must be determined by the reading of the microplate reader. All samples, washing liquid and various wastes should be treated as infectious agents. The components of different batches of this reagent shall not be mixed. 10. If there is any difference with the English manual, the English manual shall prevail. Calculation: Taking the concentration of the standard as the abscissa and the OD as the ordinate, draw a standard curve on the graph paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; multiply by the dilution factor; or use the standard The linear regression equation of the standard curve is calculated by the concentration and OD value of the sample, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor, which is the actual concentration of the sample. (This picture is for reference only) Kit performance: 1. The linear regression of the sample and the expected concentration correlation coefficient R value is more than 0.92. 2. The batch and batch see should be less than 9% and 15% respectively. Storage conditions and expiration date: 1. Kit storage :; 2-8 ℃. 2. Validity: 6 months

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